Bioprospection and molecular phylogeny of culturable endophytic fungi associated with yellow passion fruit

Endophytic microorganisms live inside the plants without causing any damage to their hosts. In the agricultural field, these endophytes might be a strategy of biological control for phytopathogens. We aimed to isolate endophytic fungi from yellow passion fruit (Passiflora edulis) leaves, evaluating its biocontrol capacity by in vitro antagonism against phytopathogen Colletotrichum sp. CNPU378. We also carried out greenhouse experiments in bean seedlings. A high colonization frequency was obtained (89%), and the molecular identification based on DNA sequencing attested Colletotrichum as the most frequent genus and minor occurrence of Curvularia endophytes. The endophytes tested showed different types of competitive interactions in in vitro antagonism inhibition rate ranging from 28.8 to 48.8%. There were 10 promising antagonists tested for their antagonist activity of crude extracts of secondary metabolites, in which strain PE-36 (20.8%) stood out among the other strains evaluated. In the greenhouse assay, plants inoculated only with endophyte Colletotrichum sp. PE-36 was symptomless and suggest that the endophyte strengthened the growth promotion in common bean plants, especially in the root length and number of leaves when compared to control plants and other treatments. Despite many fungi of Colletotrichum genus being described as causative agents of anthracnose, in this study, the plant sampled was colonized predominantly by Colletotrichum endophytes living in asymptomatic relationship. By the way, we come across a Colletotrichum sp. endophyte able to antagonize a Colletotrichum sp. pathogen.

Colletotrichum phytopathogens genus affects practically all crops grown worldwide, which are likely to be affected by one or more species of the genus (Dean et al., 2012). The common bean is one of the greatest crops in the world and is a food legume for human consumption (Talaat, 2019). Several classes of pesticides have been used annually to maintain high agricultural production. Nevertheless, the high use of these agrochemicals causes serious environmental damage (Ying, 2018), making it necessary to develop strategies that prioritize sustainable agriculture to mitigate the adverse effects of these xenobiotic chemical defenses. The formulation and sale of these agrochemicals have had a significant increase per year (Carvalho, 2017). As an alternative control, we can mention the use of microorganisms capable of producing antifungal substances or antagonizing phytopathogens (Talaat, 2019).
In this context, the current study aimed to isolate endophytic fungi from yellow passion fruit (Passiflora edulis) and to select strains with biological control potential, capable of antagonizing the phytopathogen Colletotrichum sp. CNPUV378 in in vitro conditions. We also carried out greenhouse experiments with the best endophyte in bean seedlings.

Isolation of foliar endophytic fungi
Healthy leaves of P. edulis were randomly collected in the municipality of Planaltina do Paraná, Paraná State, southern Brazil (latitude: 23º00'24''S, longitude: 52º54'50''W) and transferred to hermetic sealing plastic bags and stored at 4ºC for 24 hours. The endophytic fungi isolation was performed as previously described by Bongiorno et al. (2016), with slight modifications. Briefly, leaves were rinsed with 70% ethanol for 1 min., surface-disinfected with sodium hypochlorite solution (1% available Cl -) for 2 min., and rinsed once in 70% ethanol (for 30 s) and twice in sterile distilled water. In a laminar airflow chamber, leaves were aseptically cut into 500 fragments (5-mm 2 area). There were five foliar fragments placed on each PDA dish (Potato Dextrose Agar, pH 6.6) (Himedia, Mumbai, India) supplemented with 50 µg.mL -1 tetracycline (Sigma, St. Louis, MO) and they remained incubated at 28ºC for 7 days. The determination of colonization frequency (%) and fungal purification followed the protocols described by Ribeiro et al. (2018).

In vitro antagonism and competitive interactions between endophytes and Colletotrichum sp. CNPUV378
There were 43 endophytic isolates randomly selected and tested for their biocontrol capacity against the pathogen Colletotrichum sp. CNPUV378 (provided by EMBRAPA Grape and Wine, Bento Gonçalves, RS, Brazil). For this, the dual culture method of Campanile, Ruscelli, and Luisi (2007) modified by Polonio et al. (2015) was used. All assay was performed in triplicate.
The percentage inhibition rate of mycelial growth (IP%) was calculated following Polonio et al. (2015): IP% = (1 -MT MC -1 ) x 100, where IP% = inhibition index percentage of mycelial growth, MT = mean of triplicate area measured for treatment in cm 2 , and MC = average of triplicate control area in cm 2 .
Competitive interactions (CI) between endophytes and phytopathogens were determined according to the rating scale proposed by Innocenti, Garibyan, and Badalyan (2002) and slightly modified by Ribeiro et al. (2018), which considers four main types of interactions (A, B, C and D) and their subtypes. The types are: A = inhibition of mycelial growth with contact; B = inhibition from a distance; C = endophytic growth on the pathogen without initial inhibition; and C= partial (CA1) and complete (CA2) endophytic growth on the pathogen after initial inhibition with mycelial contact; C = partial (CB1) and complete (CB2) endophytic growth on the pathogen after initial inhibition from a distance; D = partial (DA1) and complete (DA2) pathogen growth on the endophyte after initial inhibition with mycelial contact.
To evaluate the bioactivity of CEAEs against Colletotrichum sp. CNPUV378, triplicates of PDA dishes received 5 mm autoclaved filter paper plugs inoculated with 10 μL of CEAEs (10 mg.mL -1 in methanol) in an opposite position of phytopathogen plugs and remained incubated at 28 ºC for 7 days. For controls, the paper plugs received (instead of CEAEs) the fungicide Benlate® (30 mg.mL -1 ) (C1) or methanol P.A (Synth, Diadema, SP, Brazil) (C2); C3 consisted of a phytopathogen plug inoculated in only one point of the PDA dishes. The percentage inhibition rate of mycelial growth (IP%) was calculated according to Polonio et al. (2015).

Greenhouse assay with Colletotrichum sp. endophyte and Colletotrichum sp. pathogen
For the preparation of fungal inoculum, 6 mm diameter plugs of endophyte PE-36 and pathogen were macerated into microtubes containing 1.5 mL of autoclaved distilled water and then vortexed until completely homogenized. P. vulgaris seeds were treated by immersion in 70% ethanol for 1 min., surfacedisinfected with sodium hypochlorite solution (2.5% available Cl -) for 3 min. and 70% ethanol for 30 s, and finally rinsed three times in sterile distilled water. To evaluate the efficiency of surface-disinfection, aliquots (100 μL) of water from the last rinsing was spread on Petri dishes containing PDA and TSA (Tryptone Soy Agar, pH 7.3) (Himedia, Mumbai, India). Then, seeds were transferred to dishes containing cotton soaked with 20 mL of water (both of them were previously autoclaved) and incubated at 28ºC without photoperiod for 3 days. On the 3 rd day, germinated seeds were transferred to 250 mL polypropylene containers with a mixture of Mecplant® substrate + vermiculite (3:1 v v -1 ) and soil (1:1 v v -1 ). The soil was collected at Universidade Estadual de Maringá (23º24'12.18" S and 51º56'30.54" W), autoclaved at 121ºC for 120 min.
There was one germinated seed placed in each container (with prepared soil and previously moistened with water) at approximately 3 cm depth, with the radicle facing down. The treatments were: control with water (C), plants only inoculated with the endophyte (T1), plants only inoculated with the phytopathogen (T2) and plants simultaneously inoculated with the endophyte + pathogen (T3). A total of 800 μL of each fungal suspension was used with 10 plants for each treatment (one plant per container). The presence or absence of symptoms and measurement of the biometric parameters (height, number of leaves, and root length) was performed 25 days after inoculations.

Statistical analysis
Data from the in vitro antagonism and antifungal activity the IP% values were analyzed using analysis of variance and the means were compared by the Scott-Knott test (p < 0.05 was taken to indicate a statistically significant difference) using the statistical program SISVAR 5.6 (Ferreira, 2011). To greenhouse experiment, the means of biometric parameters were compared by the Tukey's test also taking p < 0.005 to indicate a statistical significant difference (Ferreira, 2011).

Isolation and molecular identification of endophytic fungi
From the 500 leaf fragments sampled from P. edulis, we obtained a colonization frequency of 89%. The efficiency of the surface-disinfection process was attested by the absence of microbial growth in the control dishes. In order to isolate strains from the endophytic community, the surface disinfection of host plant material is necessary to ensure the elimination of epiphytes. In agreement with this current study, sodium hypochlorite and alcohol solutions have been employed in endophytic isolation protocols (Rani, Sharma, Chaturvedi, & Yadav, 2017;Salazar-Cerezo, Martinez-Montienel, Cruz-Lopez, & Martinez-Contreras, 2018;Lateef et al., 2019). For a look into the properties of endophytic microorganisms, the fragmentation of plant material represents a successful methodology that demonstrates satisfactory results for isolating the endophytic community, as already reported for different host plants (Wang et al., 2015).

In vitro antagonistic and antifungal activity of endophytes against Colletotrichum sp. CNPU378
Dual culture results showed differences among the antagonistic actions, ranging from 28.8% to 48.8% of IP% against Colletotrichum sp. CNPU378. Based on the rating scale proposed by Innocenti et al. (2002) and modified by Ribeiro et al. (2018), the types of CI observed were: A = deadlock (inhibition of phytopathogen) with mycelial contact (77.2%); DA1 = partial pathogen growth on the endophyte after initial inhibition with mycelial contact (13.6%); CA1 = partial endophyte growth on the pathogen after initial deadlock with mycelial contact (4.5%), and DB1 = partial pathogen growth on the endophyte after initial deadlock at a distance (4.5%). The Scott-Knott test highlighted 15 endophytic strains as promising antagonists in comparison with the control 2 (phytopathogen plug inoculated in only one point of Petri dish). More details regarding the antagonism index and competitive interaction are shown in Table 2 and Figure 2.   Many studies have investigated the in vitro activity of endophytes against Colletotrichum pathogens. Rabha, Naglot, Sharma, Gogoi, and Veer (2014) showed that the indexes of antagonistic activity of fungal endophytes against Colletotrichum camelliae ranged from 18.33 to 36.73. The inhibition indexes (17.52 to 49.71%) obtained by Bongiorno et al. (2016.) were similar to those obtained in this study (28.8 to 48.8%). Supporting our results, literature data has already been reported on the deadlock (mutual inhibition) at mycelial contact (type A) as the most frequent in the pathogen-endophytes competitive interaction (Campanile et al., 2007;Polonio et al., 2015;Wang et al., 2015;Bongiorno et al., 2016), in which mutual fungal inhibition occurs and neither microorganism is able to overgrow the other (Innocenti et al., 2002;Oliveira et al., 2019). Table 3 describes the results obtained for the in vitro antifungal activity of CEAEs of secondary metabolites. The higher IP% (=20.8%) against the Colletotrichum sp. CNPU378 was reported for the extract (named as code CEAE PE-36 ) obtained from the endophyte PE-36. * Means of triplicates. Means followed by the same letter in column do not differ by the Scott-Knott test (p < 0.05). Control 1 = paper plug with 10 µL of fungicide Benlate® (30 mg.mL -1 ); Control 2 = paper plug with 10 µL of methanol; C3 = phytopathogen plug inoculated in only one point of Petri dish. Concentration of CEAEs = 10 µL of 10 mg.mL -1 solution of crude extracts. Landum et al. (2016) reported that extracts with non-volatile compounds, produced by endophytes from olive plants, reached up to 26.8% of inhibition rates in dual culture tests against C. acutatum, similar to rates found in this study (up to 20.8% of mycelial inhibition). Table 4 details the biometric parameters related to the vegetative growth of bean plants. Plants inoculated only with the endophyte Colletotrichum sp. PE-36 (T1) was symptomless and showed the difference for number of leaves and root length when compared to control plants (C) and other treatments (T2 and T3). Our findings suggest that the endophyte strengthened the growth promotion of the treated plants (T1). The T2 treatment presented inferior results in all biometric parameters analyzed. In addition, the T2 treatment presented whitish lesions in the root system apparently caused by the mycelial growth of Colletotrichum sp. CNPUV378, which were not found in the other treatments, suggesting that plants inoculated with endophyte + pathogen (T3), the endophyte was able to soften the propagules of the pathogen Figure 3.  Different mechanisms of in vivo biocontrol can be employed alone or together with other strategies to trigger direct or indirect responses to overcome the pathogenicity of the causal agent (Ghorbanpour, Omidvari, Dahaji, Omidavar, & Kariman, 2018). Consistent with our in vivo assay, Rodríguez et al. (2016) reporting that the application of a suspension of an endophytic fungus on necrotic leaves reduced the spore production of the pathogen after 21 days. In addition, the authors evaluated the endophytic colonization through plant height, root length, biomass, and the number of leaves. Li, Hwang, Huang and Huang (2018) described an endophyte able to carry out the suppression of Fusarium wilt of tomato plants and beefing up their growth, mainly through the absorption of nutrients.

Conclusion
Despite many fungi of the genus Colletotrichum being described as causative agents of anthracnose, in this study, the plant sampled was 89% colonized predominantly by Colletotrichum endophytes living in asymptomatic relationship with the host Passiflora edulis, especially the isolate Colletotrichum sp. PE-36 with a highlight in in vitro and in vivo experiments.
In this work, a Colletotrichum endophytic fungus highlighted in antagonistic activity against a pathogen in the agricultural system, reinforcing the idea that endophytic microorganisms are a possible tool for the development of more sustainable, eco-friendly and easy field application as an inoculant. agricultural.